Radioimmunoassay (RIA) and related techniques are based on the principle that a substance such as an antigen, present in, e.g., a biological sample can react specifically and uniquely with another substance, such as an antibody, to form a complex that can readily be separated from either of the parent compounds. Present radioimmunoassay and related techniques all require that either the specific antibodies or the antigens be labelled with a radioactive agent usually I-125. Such labeling process may induce chemical and immunological changes, and because I-125 has a half-life of 60 days, also limits the shelf-life of the labeled product. The present invention provides a method that eliminates or minimizes the major problems of current radioactive usage. These problems are: (a) degradation of the antibody or antigen upon labeling; (b) limitation of the shelf life; and (c) radioactive waste generation. The present invention virtually eliminates the problems of radioactive waste created by the usage of the current radiolabeled materials. This invention does however retain and enhance the advantageous features of radionuclide usage.
Immunoassays require an antigen-antibody reaction, followed by a separation of the bound antigen-antibody complex from the unreacted reagent. The general method in use utilizes a second precipitating antibody, generated against the first antibody. The first antibody is used to neutralize (i.e., react to form antigen-antibody complex) the antigen. Either of these antibodies are generally produced in a rabbit or a similar animal (guinea pig, goat, sheep, etc.). Using the rabbit as an example, the first antibody produced in the rabbit is an antigen in the system of another animal, e.g., goat. Thus, when the immunoglobulin, the class of proteins which form the antibody, is injected into a goat under the proper protocol for antibody production, the immune system of the goat will consider the rabbit immunoglobin as an antigen. The new (second) antibody produced by the goat to the rabbit immunoglobin will react with the rabbit (first) antibody. This reaction with the rabbit immunoglobin of antibody makes the goat antibody a universal reagent for the reaction because while the first antibody is specific and selective for a given antigen, the second antibody recognizes the "back" part of the first antibody, which is general.
The present invention identifies a specific reaction which will allow the analyst to utilize effectively and very efficiently a specially prepared second antibody as a universal reagent. This expedient utilizes coupling of a metal binding moiety or compound such as the chelating protein, e.g., transferrin, desferoxamine, D-penicillamine or the like to the non-reactive portion of the second antibody. After the initial antigen-antibody reaction, the resultant complex is separated from excess antibody and the complex is then reacted with the second antibody that has been modified by attachment of the metal binding component. Following this step, a short-lived radionuclide, such as In-113m, is added to the bound fraction. The transferrin moiety will bind this radioactive metal with a high affinity.
The radioindium is prepared by known techniques using a generator. The half-life of this isotope is so short (100 min.) that within two days (14 half-lives) the amount of radioactivity remaining is almost nil (0.005%). However, the life of this isotope is sufficiently long so that prompt measurement of the radioactivity will enable the analyst to obtain accurate results. Other short-lived radionuclides, arbitrarily defined here to have half-lives of on the order of less than or equal to 24 hours, are also suitable.
Thus, features of the present invention include (a) the use of a short lived radioactive nuclide which enhances and increases the sensitivity of detection of substances and eliminates problems of waste disposal; (b) the use of a terminal labeling procedure which eliminates the limited shelf-life of radioimmunoassay reagents; (c) the avoidance of iodination which eliminates the chemical degradation of the antigen or antibody to be labeled; and (d) the use of a terminally labelled, "universal reagent" which markedly decreases the cost of analysis.
Immunoradiometric assay (IRMA) to determine the amount of specific antigen in a sample containing an unknown amount of the antigen is, of course, known, for example as described by Miles, Properties, Variants and Applications of the Immunoradiometric Method, La Ricerca in Clinica e in Labortorio, Vol. V. p. 59 (1975), the disclosure of which is hereby incorporated by reference. The Miles technique generally consists of reacting the unknown amount of an antigen with a soluble antibody specific to the antigen and also tagged with a radionuclide, usually I-125. Enough of the tagged soluble antibody is added to react with all of the unknown amount of the antigen being measured. Unused tagged, soluble antibody is removed by reacting it with solid-phase antigen. The amount of radioactivity remaining in the solution is a measure of the unknown amount of the antigen being measured.
An alternative IRMA method involves the preliminary insolubilization of an unknown amount of an antigen being measured and then reaction with a soluble radioactively labeled antibody. The labeled complex is thus insoluble and the unreacted labeled antibody is washed away leaving the labeled complex, with the measure of radioactivity in this solid phase complex being a measure of the amount of antigen being measured. Various methods exist for insolubilization of the antigen for purposes of carrying out this alternate IRMA method, including reaction with solid phase antibody to form an antigen-immuno-adsorbent complex (AG-ImAd), also known as, "2-site IRMA". This, however, is restricted to antigens which can either bind simultaneously to at least two antibodies or bind to the labeled antibody after the non-immunological insolubilization procedure. The technique also requires an immunoadsorbent containing highly purified antigens for preparation of the labeled antibodies.
The Miles procedure for labeling an antibody for use in an IRMA or RIA process is complicated, expensive and because of the use of I-125 has the detrimental effects initially referred to above, i.e., the possibility of altering the immunological reactivity of the labeled antibody. Further, with radionuclides such as I-125, having long half-lives, there exists a radioactive waste problem. Further, the labeled antibody used in the procedure must be highly purified to make the procedure specific to the antigen of unknown amount being measured. This is costly and requires either storage of numerous specific radioactively tagged antibodies or tagging of a specific purified antibody with a radionuclide immediately prior to the IRMA procedure. Thus, there are problems of limited shelf life in the former case and of radioactive materials controls and waste in the latter case. For highly accurate results, Miles suggests an additional refinement of the IRMA procedure in which the labeled antibody is univalent, such as produced by papain digestion, thereby adding to processing costs. Identical problems exist in the alternative 2-site IRMA procedure. In addition, further purified antibody may be necessary to form the solid phase antibody.
Miles, supra describes the adaption of both IRMA and 2-site IRMA to the use of labeled antibody (e.g., I-125-anti-IgG) as an additional "universal reagent", thereby avoiding the necessity for the preparation of radioactive antibodies specific for the unknown antigen. For example in tagging the 2-site IRMA with I-125 anti-(IgG) "universal reagent," an unknown amount of antigen, e.g., human ferritin [h(ferritin)] is reacted with solid-phase antibody, e.g., guinea-pig-anti-(ferritin) [GP-anti-(ferritin)] resulting in an insolubilized GP-anti-(ferritin)-h(ferritin) complex. The complex is then reacted with a purified specific non-radioactive antibody from a different species, e.g., Rabbit-anti-(ferritin) [R-anti-(ferritin)] resulting in an insolubilized GP-anti-(ferritin)-h(ferritin)-R-anti-(ferritin) complex. A radioactively tagged antibody of the same species as the solid-phase antibody, e.g., I-125-GP-anti(R-IgG) is then reacted with insolubilized GP-anti-(ferritin)-h(ferritin)-R-anti-ferritin), with the measure of radioactivity present on the solid-phase, once separated out, being a measure of the unknown amount of the antigen being measured.
Miles' "universal reagent", I-125-GP-anti-(R-IgG), is extremely costly to make since up to 80% of the purified GP-anti-(R-IgG) is lost during the iodination procedure. Also this reagent has a limited shelf life and the use of this reagent, as well as any others tagged with heavy metal radionuclides having long half-lives, creates significant radioactive waste disposal problems.
It has also been suggested to tag antibodies for the assay of antigen levels with radionuclides having short half-lives and susceptible of chelating with an antibody-chelate conjugate. See, Pritchard Ackerman, Tubis and Blahd, Indium-111-Labelled Antibody Heavy Metal Chelate Conjugates: A Potential Alternative to Radioiodination, Proceedings of the Society for Experimental Biology and Medicine, Vol. 151, p. 297 (1976), the disclosure of which is hereby incorporated by reference. This reference describes the possibility of conjugating the IgG molecule to compounds, e.g., transferrin, D-penicillamine and desferoxamine containing free amino groups capable of chelating heavy metal ions and binding a radionuclide, e.g., Indium-111 or iron-59 to the IgG-chelate conjugate for tagging without significantly altering the immunoreactions of the IgG-chelate conjugate.
The Pritchard, et al conjugation procedure utilizes glutaraldehyde with its two active sites to couple the free amino groups on both the chelating and the IgG molecules. Lyophilized IgG is reconstituted in physiological buffered saline (PBS) and transferrin, D-penicillamine or desferoxamine are each reconstituted in PBS. The conjugation is performed by mixing IgG, chelating compound (transferrin, D-penicillamine or desferoxamine) and glutaraldehyde in specified amounts and dialyzing to remove the unreacted glutaraldehyde and centrifuging to remove any precipitate. The In-111-IgG-chelate conjugate is then synthesized using In-111-chloride. Binding of the In-111 by the chelate portion of the IgG-chelate conjugate is described as almost instantaneous.
Pritchard, et al's proposed IgG-chelate conjugate labeling is described as overcoming some objections to the use of radioiodine; the conjugation procedure is relatively simple, the labeled conjugate is pre-prepared, the basic reagents can be incorporated in "kit" form widely useable in nuclear medicine and radioassay laboratories, and the labeling with a radionuclide, e.g., In-111 can be carried out simply and instantaneously whenever needed.
However, there are several problems existing in this prior art, Miles' disclosure of a "universal" reagent In-125-GP-anti-(R-IgG) is universal only in the sense that only one labeled antibody I-125-GP-anti-(R-IgG) need by prepared. For best results in terms of accuracy, a supply of highly purified antibody must be available for making the solid phase antibody specific to the antigen being tested, or a supply of specific solid-phase antibodies must be maintained. In addition, a highly purified supply of the antibody of the second species to the antigen must be maintained. Of course, use of .sup.125 I as a labeling radionuclide has all of the attendant problems noted above.
While the conjugate produced by Pritchard et al was a useful first step, it did not provide a basis for successful practice nor produce a general method utilizing multiple antibody moieties.
Another approach involves the formation of an antigen-antibody complex linked to an enzyme. The development of enzyme-linked immunoadsorbent assays (ELISA) has been made possible by current developments in protein conjugation techniques. Prior workers have found that the bifunctional reagent of choice for coupling enzyme labels to protein in the ELISA methods is almost exclusively glutaraldehyde. The conjugates prepared with glutaraldehyde have been reported to retain immunologic and enzymatic activity much better than those obtained with a carbodiimide coupling reagent. However, glutaraldehyde reactions with proteins have been reported to produce different degrees of crosslinkage depending on the conditions. For example, protein polymers of low molecular weight are reported to be produced at low protein concentrations, while higher protein concentrations yield water-insoluble polymers of excellent immunadsorbent capacity. A major problem of the prior art has been to retain the antigenic activity of the conjugate. Thus, for example, alkaline phosphatase-rabbit IgG conjugates, conjugated with glutaraldehyde, which contain 40% of the total protein after the coupling reaction, have been reported to produce a high degree of sensitivity in enzyme-linked immunadsorbent assays. While enzymatic activity is well preserved following such a conjugation, antigenic activity of the IgG moiety has been found to be very low, though delayed addition of the IgG to the alkaline phosphatase-glutaraldehyde reaction mixture has been reported to decrease this loss of immunological activity somewhat.
The problems enumerated in the foregoing are not intended to be exhaustive, but rather, are among many which tend to impair the effectiveness of previously suggested terminal radionuclide labeling or chelate-short lived radionuclide labeling techniques. Other noteworthy problems may also exist; however, those presented above should be sufficient to demonstrate that terminal labeling ratioimmunoassaying techniques of the prior art have not been entirely successful.
Recognizing the need for an improved terminal labeling radioimmunoassaying technique and method and an improved method for synthesizing a chelate-conjugate for use therein, it is therefore, a general feature of the present invention to provide a novel terminal labeling radioimmunoassaying method and method of synthesizing chelate-conjugates for use therein which minimizes or reduces the problems of the type previously noted.
It is a more particular feature of the present invention to provide a universal reagent having a chelating moiety for subsequent binding with a short-lived radionuclide for radioimmunoassay.
It is yet another feature of the present invention to provide a method for synthesizing an improved conjugate having a coupling moiety of a component capable of binding with specific metal ions for use in the radioimmunoassaying technique of the present invention.
Specifically, the present invention comprises an improvement to prior methods in which a "universal" antibody has been used. In accordance herewith the universal antibody is formed with a component, e.g., a chelating protein such as, e.g., transferrin, desferoxamine, or the like capable of binding a radionuclide. The second complex is reacted with a radionuclide and the radioactivity of the bound radionuclide is measured.
More specifically, the present invention relates to an immunoradiometric assaying method employing terminal labeling with a radionuclide having a relatively short half-life through the use of a chelating moiety on the protein desired to be labeled, to which the radionuclide attaches allowing measurement of the amount of the labeled protein, e.g., an antibody or antigen-antibody complex. An unknown quantity of an antigen, for example, is reacted with an insoluble antibody specific to the antigen and of a first species and the complex thus formed reacted with a second antibody specific to the antigen and of the second species. The complex thus formed is reacted with a third antibody of a species different from the second antibody and containing a chelating moiety. A suitable radionuclide having a short half-life, e.g., In-133m (which is a metastable state of Indium 113 which decays to ground level of Indium 113 by gamma emission) is then added to the complex thus formed and attaches to, i.e., "labels" the complex thus formed for purpose of radioimmunoassay.